Modified bacterial protein expression system

ABSTRACT

The present disclosure provides host cells for reliable, high yield recombinant protein production, including unstable proteins. The present host cell (e.g., a bacterial cell) is deficient in at least one protease (or a subunit of a protease) such as Clp or ClpP. The host cell may also contain an expression vector that encodes a protein or polypeptide for overexpression.

CROSS REFERENCE TO RELATED APPLICATION

This is a divisional application under 35 U.S.C. § 121 of U.S. Ser. No.16/886,156, filed May 28, 2020, which is a divisional application under35 U.S.C. § 121 of U.S. Ser. No. 15/764,070, filed Mar. 28, 2018, nowU.S. Pat. No. 10,745,730, which was the National Stage application under35 U.S.C. § 371 of International Application No. PCT/US2016/54130, filedSep. 28, 2016, which claims benefit under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 62/233,507, filed Sep. 28, 2015, which U.S.Provisional Application is incorporated by reference herein in itsentirety.

GOVERNMENT LICENSE RIGHTS

This invention was made with government support under the NationalInstitutes of Health (NIH) Grant No. GM037219. The government may havecertain rights in the invention.

BACKGROUND OF THE INVENTION Field of the Invention

The present disclosure relates to methods and systems forexpressing/overexpressing proteins in prokaryotic cells. In particular,the present disclosure relates to bacteria that have high levels ofprotein expression due to disrupted proteases.

Background Information

Commonly used protein expression systems include those derived frombacteria, yeast, baculovirus/insect cells, and mammalian cells.Bacterial expression systems, such as Escherichia coli, have theadvantage of fast growth kinetics, easily achieving high cell densitycultures, and fast and easy transformation with exogenous DNA. However,current bacterial expression systems often result in low protein yieldsor unstable proteins.

Energy-dependent degradation in Escherichia coli is carried out by anumber of ATP-dependent proteases, including Lon, FtsH, and differentClp proteases. Gottesman S., (1996) Proteases and their targets inEscherichia coli. Annu. Rev. Genet. 30:465-506. The Clp proteases arecomposed of two different multimeric components. The smaller subunit isa peptidase, either ClpP or ClpQ (HslV). Maurizi et al., (1990) Sequenceand structure of ClpP, the proteolytic component of the ATP-dependentClp protease of Escherichia coli. J. Biol. Chem. 265:12536-12545.Missiakas et al., Identification and characterization of HslV HslU (ClpQClpY) proteins involved in overall proteolysis of misfolded proteins inEscherichia coli. EMBO J. 15:6899-6909. The larger subunit is an ATPase,ClpA, ClpX, or ClpY (also called HslU). Katayama-Fujimura et all, (1987)A multiple-component ATP-dependent protease from Escherichia coli. J.Biol. Chem. 262:4477-4485. Gottesman et al., (1993) ClpX, an alternativesubunit for the ATP-dependent Clp protease of Escherichia coli. J. Biol.Chem. 268:22618-22626. Rohrwild et al., (1996) HslV-HslU: a novelATP-dependent protease complex in Escherichia coli related to theeukaryotic proteasome. Proc. Natl. Acad. Sci. USA 93:5808-5813. Wu, etal., Redundant In Vivo Proteolytic Activities of Escherichia coli Lonand the ClpYQ (HslUV) Protease, J. Bacteriol. June 1999 vol. 181 no. 123681-3687.

Either ClpA or ClpX can associate with ClpP to form an ATP-dependentprotease (ClpAP or ClpXP). ClpY associates with ClpQ to form an active,energy-dependent protease. While ClpY and ClpX show significant sequencesimilarity to each other and to ClpA, ClpP and ClpQ(HslV) are notrelated at the sequence level. ClpP is a serine protease, found in manyprokaryotes and in the organelles of many eukaryotes; ClpQ has acatalytic amino-terminal threonine residue and shows sequence similarityto the eukaryotic proteasome P subunit. Seemuller et al., (1995)Proteasome from Thermoplasma acidophilum: a threonine protease. Science,268:579-582.

Like other parental E. coli B strains, BL21 cells are deficient in theLon protease, which degrades many foreign proteins. Gottesman S. (1996)Proteases and their targets in Escherichia coli. Annu. Rev. Genet. 30465-506. Another gene missing from the genome of the ancestors of BL21is the outer membrane protease OmpT, whose function is to degradeextracellular proteins. In the BL21(DE3) strain, the λDE3 prophage wasinserted in the chromosome of BL21 and contains the T7 RNAP gene underthe lacUV5 promoter. The BL21(DE3) and its derivatives, as well as theK-12 lineage are widely used for protein expression. Rosano et al.,Recombinant protein expression in Escherichia coli: advances andchallenges, Front Microbiol. 2014, 5:172.

The present disclosure relates to bacteria that have high levels ofprotein expression due to disruption of at least one protease. Thepresent disclosure also provides for a method to improve the yield ofexpressed recombinant proteins, including unstable proteins.

SUMMARY

The present disclosure provides for an engineered bacterium comprisingat least one deficient protease. The protease may be Clp, ClpP, ClpQ(HslV), ClpAP, ClpXP, ClpAXP, ClpYQ, ClpA, ClpX, ClpY (HslU), orcombinations thereof. The bacterium may overexpress a polypeptide (orprotein) at a level which is at least about 2 fold, at least about 3fold, at least about 4 fold, at least about 5 fold, at least about 6fold, at least about 8 fold, at least about 10 fold, at least about 12fold, at least about 14 fold, at least about 15 fold, at least about 16fold, at least about 18 fold, at least about 19 fold, at least about 20fold, at least about 25 fold, or at least about 30 fold, of the level ofthe polypeptide (or protein) produced by a bacterium not engineered withthe deficient protease.

The present disclosure also provides for an engineered bacteriumcomprising a total or partial deletion of at least one gene encoding aprotease. The protease may be Clp, ClpP, ClpQ (HslV), ClpAP, ClpXP,ClpAXP, ClpYQ, ClpA, ClpX, ClpY (HslU), or combinations thereof.

Also encompassed by the present disclosure is a method foroverexpressing a polypeptide (or protein). The method may comprise thefollowing steps: (a) culturing the present engineered bacterium toproduce the polypeptide (or protein); and (b) isolating the polypeptide(or protein).

The present disclosure also provides for a method for overexpressing apolypeptide (or protein) in bacteria, the method comprising the stepsof: (a) transforming the present engineered bacterium with a nucleicacid sequence encoding the polypeptide (or protein); (b) culturing thebacterium to produce the polypeptide (or protein); and (c) isolating thepolypeptide (or protein).

The present disclosure also provides for a method for expressing apolypeptide (or protein) in bacteria, the method comprising the stepsof: (a) engineering a bacterial cell to decrease level and/or activityof at least one protease, wherein the protease is Clp, ClpP, ClpQ(HslV), ClpAP, ClpXP, ClpAXP, ClpYQ, ClpA, ClpX, ClpY (HslU), orcombinations thereof; (b) transforming the engineered bacterial cellwith a nucleic acid sequence encoding the polypeptide; (c) culturing thebacterium to produce the polypeptide; and (d) isolating the polypeptide.

The engineered bacterium may have deficient ClpP. The engineeredbacterium may have a total or partial deletion of a gene encoding ClpP.

The engineered bacterium may further comprise deficient Lon, OmpT, FtsH,or combinations thereof. The engineered bacterium may further comprisedeficient Lon and deficient OmpT.

The gene encoding the protease may be knocked out or knocked down in theengineered bacterium. The gene encoding the protease may be mutated ordeleted in the engineered bacterium.

The protease may be deleted by replacing the protease gene with aselection marker, such as an antibiotic resistance gene. For example,the antibiotic may be kanamycin, chloramphenicol, tetracyclin,ampicillin, vancomycin or erythromycin. In one embodiment, theantibiotic is kanamycin.

The bacterium may belong to the Escherichia, Methylomonas,Methylobacter, Methylococcus, Methylosinus, Salmonella, Erwina,Haematococcus, Rhodobacter, Myxococcus, Corynebacteria, Pseudomonas orBacillus genus. In one embodiment, the bacterium is Escherichia coli.For example, the Escherichia coli strain may be E. coli BL21 (DE3) or E.coli BL21.

The engineered bacterium may further comprise a nucleic acid sequenceencoding a polypeptide for overexpression. The polypeptide may beheterologous or homologous to the bacterium.

The polypeptide may be an enzyme, a growth factor, a blood clottingfactor, a hormone, or a transcription factor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a chromatogram of purification of SUMO-NunΔN (SUMO-NunDN)using the HiTrap IMAC column (GE Healthcare Life Sciences). The columnwas eluted with gradient 20%-100% Ni-B buffer over 12 CV (columnvolumes). 5-ml fractions were collected.

FIG. 1B shows SDS-PAGE analysis of samples of different fractions fromthe HiTrap IMAC column. Fractions 8-13 were collected for digestion byUlp-1. Lane 1: Supernatant; Lane 2: Flow-through; Lane 3: 4% Ni-B wash;Lane 4: 8% Ni-B wash; Lane 5: 16% Ni-B wash; Lane 6: elution in a 10-mlfraction; Lanes. 7-14: 5-ml eluted fractions. The arrow points to theSUMO-NunDN band on the SDS-PAGE.

FIG. 2A shows a chromatogram of purification of NunΔN (NunDN) using theMono S column (GE Healthcare Life Sciences) after SUMO is cleaved. Thecolumn was eluted with gradient 10-100% MonoS-B buffer over 18 CV.

FIG. 2B shows SDS-PAGE analysis of different samples. Lane 1: beforeSUMO cleavage; Lane 2: after SUMO cleavage; Lane 3: supernatant fromcentrifugation of the sample after SUMO cleavage reaction; Lane 4:pellet from centrifugation of the sample after SUMO cleavage reaction;Lane 5: flow-through of the Mono S column; Lane 6-1: 4-ml elutedfractions (corresponding to the fractions marked by the two horizontalbars in FIG. 2A).

FIG. 3A shows a chromatogram of purification of SUMO-NunDN using theHiLoad SuperDex 75 column (GE Healthcare Life Sciences).

FIG. 3B shows SDS-PAGE analysis of samples of different fractions fromthe HiLoad SuperDex 75 column. Lane 1: input sample; Lanes 2-8: elutedfractions as indicated in FIG. 3A. Lanes 2-4 correspond to 10-mlfractions; Lanes 5-8 correspond to 5-ml fractions.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides host cells for reliable, high yieldrecombinant protein production, including unstable proteins. The presentbacterial cells are able to produce (e.g., overexpress) higher levels ofproteins (or polypeptides), compared to commonly used strains, such asE. coli BL21 (DE3). The proteins (or polypeptides) may be heterologousor homologous to the host cells. The present host cell (e.g., abacterial cell) is deficient in at least one protease (or a subunit of aprotease) such as Clp or ClpP. The host cell may also contain anexpression vector that encodes a protein or polypeptide for production(e.g., overexpression).

The present host cells may be deficient in one or more of the followingproteases (or subunits of protease): Clp, ClpP, ClpQ (HslV), ClpAP,ClpXP, ClpAXP, ClpYQ, ClpA, ClpX, ClpY (HslU), and combinations thereof.

The ClpP protease may be disrupted (mutated or deleted) in the hostcell. In addition to ClpP, other protease(s) may also be disrupted. Inone embodiment, the host cell (e.g., a bacterium) has at least ClpP andthe Lon protease disrupted. In another embodiment, the host cell (e.g.,a bacterium) has at least ClpP and the OmpT protease disrupted. In athird embodiment, the host cell (e.g., a bacterium) has at least ClpP,Lon and OmpT disrupted.

The expression level of a target protein in the present engineered hostcell (with a disrupted protease or a subunit of a protease, e.g., ClpPor others as described herein) may be greater than about 1.1 fold,greater than about 1.2 fold, greater than about 1.3 fold, greater thanabout 1.4 fold, greater than about 1.5 fold, greater than about 1.6fold, greater than about 1.7 fold, greater than about 1.8 fold, greaterthan about 1.9 fold, greater than about 2 fold, greater than about 3fold, greater than about 4 fold, greater than about 5 fold, greater thanabout 6 fold, greater than about 8 fold, greater than about 10 fold,greater than about 12 fold, greater than about 14 fold, greater thanabout 15 fold, greater than about 16 fold, greater than about 18 fold,greater than about 19 fold, greater than about 20 fold, greater thanabout 25 fold, or greater than about 30 fold, of the expression level ofthe target protein in an unmodified host cell (with intact protease or asubunit of a protease) or a host cell not engineered with the deficientprotease (or a subunit of a protease).

The present disclosure provides a method of producing a desiredpolypeptide in host cells deficient in at least one protease (or asubunit of a protease). The method may contain the following steps: (a)culturing the host cells in a culture medium to allow expression of thedesired polypeptide in the host cells; and (b) isolating the desiredpolypeptide from the host cells.

In some embodiments, the present method comprises introducing anexpression vector encoding a polypeptide (protein) into a host cell(e.g., a bacterial cell), thereby expressing the polypeptide.

Also encompassed by the present disclosure is a method for expressing(or overexpressing) a polypeptide (protein) in host cells. The methodmay contain the following steps: (a) transforming the engineered hostcell (e.g., a bacterial cell) with a nucleic acid sequence encoding apolypeptide (protein), where the engineered host cell has at least onedeficient protease (or a subunit of a protease); (b) culturing the hostcell to produce the polypeptide (protein); and (c) isolating thepolypeptide (protein).

The present disclosure provides a method of expressing a polypeptide(protein) in a host cell (e.g., a bacterial cell). The method maycontain the following steps: (a) engineering a host cell to decreaselevel and/or activity of a protease (or a subunit of a protease); (b)transforming the engineered host cell with a nucleic acid sequenceencoding a polypeptide (protein); (c) culturing the bacterium to producethe polypeptide (protein); and (d) isolating the polypeptide (protein).the context clearly dictates otherwise. Thus, for example, references to“a nucleic acid” includes one or more nucleic acids, and/or compositionsof the type described herein which will become apparent to those personsskilled in the art upon reading this disclosure and so forth.

Proteases

In the present host cell, a protease (or a subunit of a protease) may bedeficient or disrupted (mutated or deleted). The level and/or activityof the protease (or a subunit of a protease) in the present host cell isdecreased compared to the cell which has the wild-type protease or thewild-type protease subunit. For example, an ATPase submit of a proteasemay be deficient. The protease or a subunit of a protease that may bedeficient in the present host cells include, but are not limited to, aClp protease, ClpP, ClpQ (HslV), ClpAP, ClpXP, ClpAXP, ClpYQ (HslUV),Lon, OmpT, FtsH (HflB), ClpA, ClpX, ClpY (HslU), or combinationsthereof.

The present host cell may also be deficient in an ortholog or a homologof the protease or (a subunit of a protease) as described herein.

One or more than one protease (or a subunit of a protease) may bedisrupted in the present host cell, including 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or more proteases (or subunits of protease).

In addition to ClpP, other protease(s) may also be disrupted. In oneembodiment, the bacteria have at least ClpP and the Lon proteasedisrupted. In another embodiment, the bacteria have at least ClpP andthe OmpT protease disrupted. In a third embodiment, the bacteria have atleast ClpP, Lon and OmpT disrupted.

The protease may be an ATP-dependent protease. The protease may be aserine protease.

The term “disruption” or “attenuation” means reducing or suppressing theactivity or level of the protease (or a subunit of a protease) in thehost cell.

The protease (or a subunit of a protease) may be deleted (partially orin its entirety), partially truncated, or mutated. The gene encoding theprotease (or a subunit of a protease) may be deleted (partially or inits entirety), partially truncated, or mutated. The mutation in theprotease may be a mutation of any type. For instance, the mutation mayconstitute a point mutation, a frame-shift mutation, an insertion, adeletion of part or all of the protease (or a subunit of a protease). Inone embodiment, a portion of the bacterial genome encoding the protease(or a subunit of a protease) has been replaced with a polynucleotide(e.g., a heterologous polynucleotide). In another embodiment, thepromoter of the gene encoding a protease or a subunit of a protease maycontain one or more mutations. Examples of mutations include anymutation that introduces one or more stop codons within the codingregion of a gene encoding the protease (or a subunit of a protease), anymutation that modifies the structure of the protease (or a subunit of aprotease). The protease (or a subunit of a protease) may be disrupted,for example, by using a weak promoter for a gene encoding the protease(or a subunit of a protease), and/or using an allele which encodes acorresponding protease (or a subunit of a protease) which has a lowactivity or inactivates the corresponding protease (or a subunit of aprotease).

The gene encoding the protease (or a subunit of a protease) may beknocked out or knocked down.

The term “knock out” means deleting or inactivating one or more genes ina genetically engineered cell.

The term “knock down” means reducing or attenuating the expression ofone or more genes in a genetically engineered cell.

The protease (or a subunit of a protease) in the present host cells maybe engineered to have attenuated, diminished or abrogated level and/oractivity.

When a protease (or a subunit of a protease) is disrupted, the activityor level of the corresponding protease (or a subunit of a protease) maybe reduced to, e.g., 0 to 75%, 0 to 50%, 0 to 25%, 0 to 10%, or 0 to 5%of the activity or level of the wild-type protease (or a subunit of aprotease), or of the activity or level of the protease (or a subunit ofa protease) in the starting cells before the protease or a subunit of aprotease is attenuated.

The present host cell may also be deficient in another protein or DNAwhich results in a deficiency in a protease (or a subunit of aprotease). For example, the present host cell may contain any mutationin a protein and/or DNA that decreases the transcription or translationof a protease (or a subunit of a protease). The present host cell maycontain any mutation that directly or indirectly modifiespost-transcription or post-translation process of a protease (or asubunit of a protease) so that modifies its expression or activity.

In one embodiment, the present host cell contains a compound or entity,e.g., antisense RNA that decreases the transcription and/or translationof a protease (or a subunit of a protease), or decreases the activity orfunction of a protease (or a subunit of a protease). In certainembodiments, altered gene expression and/or protease activity may beaccomplished using, e.g., antisense RNA, siRNA, miRNA, ribozymes, triplestranded DNA, nucleic acids, small molecules, protease inhibitors, atransposon, and other manipulations for altered gene expression and/oractivity.

When disrupting a protease (or a subunit of a protease), the disruption(e.g., mutation, deletion, or inactivation) may be detected by aselection marker so that the modified host cells can be identified andisolated. The protease (or a subunit of a protease) may be deleted byreplacing the protease gene with a selection marker.

The selection marker may be any suitable marker gene such as anantibiotic resistance gene or an auxotrophic marker. The antibioticsthat can be used to select the host cells having the antibioticresistance genes include, but are not limited to, kanamycin,chloramphenicol, tetracyclin, ampicillin, vancomycin and erythromycin orany other representatives of the beta-lecterns, the aminoglycosides, theglycopeptides or the macrolide antibiotics.

In one embodiment, the present bacteria are constructed by P1transduction of a clpP::kan from the KEIO collection into BL21(DE3).This results in the genotype of the bacteria being BL21(DE3) plus aclpP::kan mutation (a kan insert in the clpP gene derived from the KEIOcollection). The E. coli B strains obtained may be E. coli B BL21(DE3)clpP::kan or E. coli B BL21(DE3) AclpP. In one embodiment, the bacteriamay be F-ompT hsdS_(B) (r_(B)-m_(B)-) gal dcm (DE3) clpP::kan.

Host Cells Including Bacteria

The host cell producing the protein(s) or polynucleotide(s) of interestcan be any host cell. The present host cells may be prokaryotic oreukaryotic. In some embodiments, the host cell is a bacterial cell. Insome embodiments, the host cell is a yeast cell. In some embodiments,the cells are mammalian cells. In some embodiments, the cells areantigen-presenting cells, such as dendritic cells. In some embodiments,the host cells are isolated.

The present bacteria can be from any species or strain.

Prokaryotes to use in the present systems and methods includeeubacteria, such as Gram-negative or Gram-positive organisms, forexample, Enterobacteriaceae such as Escherichia (e.g., E. coli),Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., S.typhimurium, Serratia, e.g., S. marcescans, and Shigella, as well asBacilli, Pseudomonas such as P. aeruginosa, and P. fluorescens,Ralstonia, e.g., R. eutropha, Staphylococcus, e.g. S. carnosus, andStreptomyces.

The bacterium may be in the Enterobacteriaceae family which includes,but are not limited to, bacteria belonging to the genera Escherichia,Enterobacter, Envinia, Klebsiella, Pantoea, Photorhabdus, Providencia,Salmonella, Serratia, Shigella, Geobacillus, Morganella Yersinia, etc.Specifically, those classified into the Enterobacteriaceae according tothe taxonomy used in the NCBI (National Center for BiotechnologyInformation) database can be used.

In some embodiments, the present bacterium belongs to the genusEscherichia, Bacillusm, or Pantoea.

Non-limiting examples of bacteria belonging to the genus Escherichiainclude bacteria described by Neidhardt et al. (Escherichia coli andSalmonella typhimurium, American Society for Microbiology, WashingtonD.C., 1208, Table 1).

In one embodiment, the present bacterium may be Escherichia coli (E.coli). They include, but are not limited to, E. coli B strains, K-12strains, C strains, and W strains. The bacterial strains include, butare not limited to, BL21, BL21(DE3), BL21(DE3)pLysS, BL21(DE3)pLysE, andBL21(AI). The E. coli strain B BL21 (DE3) is described in Studier etal., J. Mol. Biol. (1986) 189:113-130 and Studier et al. Methods inEnzymology (1990) 185:60-89. The E. coli strain B BL21 (DE3) iscommercially available (e.g., New England BioLabs, Stratagene andNovagen). Non-limiting examples of the E. coli K-12 strains includeAD494, Origami™ (Novagen), and HMS 174. Derman et al., (1993) Mutationsthat allow disulfide bond formation in the cytoplasm of Escherichiacoli. Science 262 1744-1747. Campbell et al., (1978) Geneticrecombination and complementation between bacteriophage T7 and clonedfragments of T7 DNA. Proc. Natl. Acad. Sci. U.S.A. 75 2276-2280.Non-limiting examples of the strains of E. coli also include, E. coli294 (ATCC 31,446), E. coli B, E. coli X1776 (ATCC 31,537), E. coli W3110 (ATCC 27,325), and CY15070 (Paluh et al., Nucl. Acids Res. 24;14(20): 7851-60 (1986); (ATCC #47022)). U.S. Pat. No. 8,535,909.

Non-limiting examples of bacteria belonging to the genus Pantoea includePantoea agglomerans, Pantoea ananatis, Pantoea stewartii and the like.

Non-limiting examples of bacteria belonging to the genus Bacillusinclude B. subtilis, B. megaterium, B. amyloliquefaceins, B.thuringiensis, B. licheniformis, B. sphericus, B. anthracis, B. cereus,B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B.amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B.coagulans, B. circulans, B. lautus, B. pumilus, B. thuringiensis etc. Asused herein, “Bacillus sp.” includes all species within the genusBacillus. In some embodiments, the Bacillus strain of interest is analkalophilic Bacillus. Numerous alkalophilic Bacillus strains are known(See e.g., Aunstrup et al., Proc IV IFS: Ferment. Tech. Today, 299-305[1972]). Numerous B. subtilis strains are known (See e.g., 1A6 (ATCC39085), 168 (1A01), SBI9, W23, Ts85, B637, PB1753 through PB1758,PB3360, JH642, 1A243 (ATCC 39,087), ATCC 21332, ATCC 6051, M1113, DE100(ATCC 39,094), GX4931, PBT 110, and PEP 211 strain; Hoch et al.,Genetics, 73:215-228 [1973]). Palva et al., Gene, 19:81-87 [1982];Fahnestock and Fischer, J. Bacteriol., 165:796-804 [1986]; and Wang etal, Gene 69:39-47 [1988].

In some embodiments, the bacteria are members of the genus Bacillus. Inanother embodiment, the bacteria are Bacillus anthracis. In stillanother embodiment, the bacteria are Yersinia pestis. In otherembodiments of the invention, the bacteria are from the genusSalmonella. In some embodiments, the bacteria are Salmonellatyphimurium. In some embodiments, the bacteria belong to the genusShigella. For instance, in some embodiments, the bacteria are Shigellaflexneri. In some embodiments, the bacteria are members of the genusBrucella. In an alternative embodiment, the bacteria are mycobacteria.The mycobacteria are optionally a member of the species Mycobacteriumtuberculosis. In some embodiments, the bacteria are BacillusCalmette-Guerin (BCG). In some embodiments, the bacteria are E. coli. Insome embodiments, the bacteria may be Listeria, Bacillus anthracis,Yersinia pestis, Salmonella, and mycobacteria.

Examples of prokaryotic host cells also include, but are not limited to,Salmonella typhimurium, Bacillus subtilis, Bacillus licheniformis,Bacillus megaterium, Bacillus brevi, Pseudomonas aeruginosa, Pseudomonasfluorescens, Ralstonia eutropha, Staphylococcus carnosus and Serratiamarcescans.

Examples of host cells also include, but are not limited to,Acinetobacter, Agrobacterium tumefaciens, Azoarcus, Bacillus anthracis,Bacillus clausii, Bacillus licheniformis, Bacillus amyloliquefaciens,Bacillus subtilis, Bacillus lentus, Bacillus halodurans, Bifidobacteriumlongum, Buchnera aphidicola, Campestris, Campylobacter jejuni,Clostridium perfringens, Escherichia coli, Erwinia carotovora,Francisella tularensis, Haemophilus influenzae, Helicobacter pylori,Mycobacterium tuberculosis, Neisseria meningitides, Pseudomonasaeruginosa, Prochlorococcus marinus, Streptococcus pneumoniae,Salmonella enterica, Shewanella oneidensis, Salmonella enterica,Salmonella typhimurium, Staphylococcus epidermidis, Staphylococcusaureus, Shigella flexneri, Streptomyces coelicolor, Streptomyceslividans, Tropheryma whipplei, Tularensis, Temecula, Thermosynechococcuselongates, Thermococcus kodakarensis, Xanthomonas axonopodis,Xanthomonas campestris; Xylella fastidiosa and Yersinia pestis hostcells.

-   The starting host cell in which a protease (or a subunit of a    protease) is then disrupted may be from a non-recombinant strain,    mutants of a naturally-occurring strain, or a recombinant strain    that is modified to be deficient in a protease.

In some embodiments, the bacteria are attenuated. In some embodiments,the bacteria are attenuated for cell-to-cell spread, entry intonon-phagocytic cells, and/or proliferation (relative to a bacteriumwithout the attenuating mutation). The bacteria may be attenuated bymutation or by other modifications.

The microorganisms prepared according to the invention can be culturedcontinuously or discontinuously in the batch process (batch culture), orin the fed batch (feed process), or repeated fed batch process(repetitive feed process) for the purpose of production of the proteinor polypeptide of interest.

The culture of the recombinant microorganisms according to the presentinvention may be carried out according to a method well known in theart. Culture conditions, including culture temperature, time and the pHof medium can be suitably controlled. The collection of recombinantmicrobial cells from the culture broth can be carried out usingconventional isolation techniques, for example, centrifugation orfiltration.

The medium used for culturing the present host cells may be either asynthetic medium or a natural medium. In one embodiment, the mediumincludes a carbon source and a nitrogen source and minerals and, ifnecessary, appropriate amounts of nutrients which the host cells requirefor growth. The culture medium to be used must meet the requirements ofthe particular strains in a suitable manner. Descriptions of culturemedia for various microorganisms are contained in Manual of Methods forGeneral Bacteriology of the American Society for Bacteriology(Washington D.C., USA, 1981). Sugars and carbohydrates, such as e.g.glucose, sucrose, lactose, fructose, maltose, molasses, starch andcellulose, oils and fats, such as e.g. soya oil, sunflower oil,groundnut oil and coconut fat, fatty acids, such as e.g. palmitic acid,stearic acid and linoleic acid, alcohols, such as e.g. glycerol andethanol, and organic acids, such as e.g. acetic acid, can be used as thesource of carbon. These substances can be used individually or as amixture. Organic nitrogen-containing compounds, such as peptones, yeastextract, meat extract, malt extract, corn steep liquor, soya bean flourand urea, or inorganic compounds, such as ammonium sulfate, ammoniumchloride, ammonium phosphate, ammonium carbonate and ammonium nitrate,can be used as the source of nitrogen. The sources of nitrogen can beused individually or as a mixture. Phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogen phosphate or the correspondingsodium-containing salts can be used as the source of phosphorus. Theculture medium may comprise salts of metals, such as e.g. magnesiumsulfate or iron sulfate. Growth substances, such as amino acids andvitamins, may be employed. Suitable precursors can moreover be added tothe culture medium. The starting substances mentioned can be added tothe culture in the form of a single batch, or can be fed in during theculture in a suitable manner.

The proteins or polypeptides to be produced in the host cells can besynthesized as fusion peptides, secreted into the media or into theperiplasmic space, produced in free form in the cytoplasm, oraccumulated in intracellular bodies such as in inclusion bodies. Thehost cell may or may not secrete the polypeptides of interest.

Purification of the proteins or polypeptides involves the use ofconventional methodologies which may be adapted by conventionaltechniques to the protein (or polypeptide) of interest.

Polypeptides/Proteins to be Expressed or Overexpressed

A “desired polypeptide (protein)”, “polypeptide (protein) of interest,”or “target polypeptide (protein)” refers to the protein/polypeptide tobe expressed (e.g., overexpressed) by the host cell.

The engineered (recombinant) bacteria may constitutively or induciblyexpress one or more of proteins/polypeptides. The gene of theprotein/polypeptides may be under the control of a constitutive orinducible promoter.

The desired polypeptide (or polypeptide of interest) may be eitherhomologous or heterologous to the host. As used herein, the term“heterologous protein” refers to a protein or polypeptide that does notnaturally occur in the host cell. Similarly, a “heterologouspolynucleotide” refers to a polynucleotide that does not naturally occurin the host cell. The target polypeptide (protein) may be a heterologouspolypeptide (protein) which cannot be normally found in transformed hostcells expressing the protein.

The desired polypeptide may be an enzyme, a transcription factor, ahormone, a growth factor, a receptor, vaccine, an antibody or itsfragment, or the like.

The desired polypeptide/protein may be an enzyme, including, but notlimited to, amylolytic enzymes, proteolytic enzymes, cellulytic enzymes,oxidoreductase enzymes, cellulose, plant wall degrading enzymes,amylases, proteases, xylanases, lipases, laccases, phenol oxidases,oxidases, cutinases, cellulases, hemicellulases, esterases,perioxidases, catalases, glucose oxidases, phytases, pectinases,glucosidases, isomerases, transferases, galactosidases and chitinases.

Proteins (or polypeptides) that may be produced (e.g., overexpressed) bythe present host cells and methods include, but are not limited to,growth factors (e.g. epidermal growth factor, insulin-like growthfactor-1); blood clotting factors (e.g. anti-hemophilic factor);hormones (e.g., insulin, glucagon, growth hormone, somatotropin,erythropoietin); cytokines (e.g., interferons, interleukins; granuloctyecolony-stimulating factor, granulocyte-macrophage colony-stimulatingfactor, CD86); chemokines (e.g., CCL3); receptors (e.g., chemokinereceptors, tyrosine kinase receptors); enzymes (e.g., proteases,lipases, carbohydrases, chymosin, DNAase, prourokinase, argininedeaminase, cytosine deaminase, L-asparaginase); enzyme activators (e.g.,tissue-type plasminogen activator); enzyme inhibitors (e.g, tissueinhibitors of metalloproteases); peptides (e.g., hirudin, neuregulin-1fragments); antibody fragments (e.g., Fab fragments); protein scaffolds(e.g. Adnectins, Affibodies, Anticalins, DARPins, engineered Kunitz-typeinhibitors, tetranectins, A-domain proteins, lipocalins, repeat proteinssuch as ankyrin repeat proteins, immunity proteins. α2p8 peptide, insectdefensin A, PDZ domains, charybdotoxins, PHD fingers, TEM-1β-lactamase,fibronectin type III domains, CTLA-4, T-cell resptors, knottins,neocarzinostatin, carbohydrate binding module 4-2, green fluorescentprotein, thioredoxin); vaccines (e.g. influenza vaccines, anthraxvaccines such as rPA vaccines, hepatitis E virus vaccines such as ORF2vaccines, human papilloma virus vaccines); toxins; and immunotoxins(Misawa and Kumagai, Biopolymers 51: 297-307 (1999); Zhang et al,Protein Expr. Purif. 25(1): 105-13 (2002); Demain, Trends in Biotech.18(1): 26-31 (2000); Gebauer & Skerra, Curr. Opin. Chem. Biol. 13:245-55(2009); Gill & Damle, Curr. Opin. Biotech 17: 653-58 (2006); Hosse etal, Protein Sci. 15:14-27 (2006); Skerra, Curr. Opin. Biotech 18:295-3-4 (2007); Song et al, PLoS ONE 3(5): e2257 (2008); Vahedi et al,Applied Biochem. and Biotech. 157(3): 554-61 (2009); Hakim and Benhar,MAbs. 1(3): 281-87 (2009)).

Proteins (or polypeptides) that may be produced by the present hostcells and methods include, but are not limited to, hGH, glucagon-likepeptides, interleukins, insulin analogs, adiponectin, FGF-21, trypsin,aprotinin, amylin, leptin and analogs thereof, enzymes such assialidase, transglutaminase (tGase), HRV (Human Rhino Virus) 3Cprotease, Tobacco Etch Virus (TEV) protease and variants thereof.

In some embodiments, the protein/polypeptide expressed by the presenthost cells is an antibody or an antibody fragment, such as an Fv, adisulfide-linked Fv, an scFv, a kappa light chain fragment, a lambdalight chain fragment, and a Fab fragment.

One or more polypeptides and/or proteins may be produced. Thepolynucleotides encoding the polypeptides/proteins may be on differentexpression vectors or may be on the same expression vector.

The polynucleotide encoding the polypeptide/protein of interest may beoperably linked to regulatory sequences, such as promoters, enhancers orone or more other transcriptional regulatory sequences, optionally aspart of a vector comprising these sequences.

Vectors

The present host cell may comprise at least one vector (e.g., a plasmid)comprising at least one nucleotide sequence encoding a (heterologous orhomologous) protein (or polypeptide). The invention also provides amethod of expression, wherein a (heterologous or homologous) protein canbe expressed in the host cells.

The vectors may contain regulatory sequences, e.g., transcriptional ortranslational control sequences required for expressing the exogenouspolypeptide. Suitable regulatory sequences include but are not limitedto replication origin, promoter, enhancer, repressor binding regions,transcription initiation sites, ribosome binding sites, translationinitiation sites, and termination sites for transcription andtranslation. One or more regulatory sequences may be operably linked tothe gene encoding the protein (or polypeptide) to be expressed in thehost cells.

The term “recombinant” when used in reference to a cell, nucleic acid,protein or vector, indicates that the cell, nucleic acid, protein orvector, has been modified by the introduction of a heterologous nucleicacid or protein or the alteration of a native nucleic acid or protein,or that the cell is derived from a cell so modified. Thus, for example,recombinant cells express nucleic acids or polypeptides that are notfound within the native (non-recombinant) form of the cell or expressnative genes that are otherwise abnormally expressed, under-expressed,overexpressed or not expressed at all. A “recombinant protein” is aprotein made using recombinant techniques, i.e., through the expressionof a recombinant nucleic acid as depicted above. As used herein,“recombinant host cells” and “engineered host cells” are usedinterchangeably; “recombinant bacterium” and “engineered bacterium” areused interchangeably.

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. A number of bacterial expression vectors areavailable commercially and through the American Type Culture Collection(ATCC), Rockville, Md.

Tags can also be added to recombinant proteins to increase proteinstability and/or provide convenient methods of isolation, e.g., SUMO,c-myc, biotin, poly-Histidine (His), etc.

A nucleic acid sequence may be introduced into a cell by transformation,transfection, transduction, or other suitable techniques. Transformationof a bacterium with a DNA coding for a target protein means introductionof the DNA into a bacterium cell to increase expression of the genecoding for the target protein.

Bacterial transformation may be referred to as genetic alteration of abacterial cell resulting from the uptake of exogenous genetic material(exogenous DNA) from its surroundings through the cell membrane. Fortransformation to occur, bacteria should be in a state of competence.Some bacteria are naturally capable of taking up DNA under laboratoryconditions. For other bacteria, Artificial competence can be induced inlaboratory procedures that involve making the cell passively permeableto DNA by exposing it to conditions that do not normally occur innature. In one embodiment, chilling cells in the presence of divalentcations (such as Ca²⁺, Mg²⁺) prepares the cell walls to become permeableto plasmid DNA. Cells are incubated on ice with the DNA and then brieflyheat shocked. Electroporation is another approach to promote competenceof the bacterial cells, where the cells are briefly shocked with anelectric field. Green & Sambrook, Molecular Cloning, A Laboratory Manual(4th edition, 2012); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994).

Increased Protein/Polypeptide Production

The present host cell, with disrupted protease (or a subunit of aprotease), produces a target protein in an amount that is greatercompared to a cell in which the protease (or a subunit of a protease)has not been disrupted.

The expression level of the target protein in the present host cell(with disrupted protease or a subunit of a protease, e.g., ClpP orothers as described herein) may be greater than about 1.1 fold, greaterthan about 1.2 fold, greater than about 1.3 fold, greater than about 1.4fold, greater than about 1.5 fold, greater than about 1.6 fold, greaterthan about 1.7 fold, greater than about 1.8 fold, greater than about 1.9fold, greater than about 2 fold, greater than about 3 fold, greater thanabout 4 fold, greater than about 5 fold, greater than about 6 fold,greater than about 8 fold, greater than about 10 fold, greater thanabout 12 fold, greater than about 14 fold, greater than about 15 fold,greater than about 16 fold, greater than about 18 fold, greater thanabout 19 fold, greater than about 20 fold, greater than about 25 fold,greater than about 30 fold, about 2 fold-10 fold, about 2 fold-5 fold,about 1.5 fold-8 fold, about 2 fold-8 fold, about 5 fold-15 fold, about5 fold-20 fold, about 2 fold-30 fold, about 2 fold-20 fold, about 5fold-20 fold, or about 5 fold-10 fold, of the expression level of thetarget protein in an unmodified host cells (with intact or undisruptedprotease or a subunit of a protease) or a host cell not engineered withthe deficient protease (or a subunit of a protease).

The expression level of the target protein in the present bacteria (withdisrupted protease or a subunit of a protease, e.g., ClpP or others asdescribed herein) may be increased by at least 20%, about 30%, about40%, about 50%, about 60%, about 70%, about 75% about 80%, about 85%,about 90%, about 95%, about 100%, about 125%, about 150%, about 175%,about 200% or about 250% as compared to the expression level of thetarget protein in the unmodified host bacteria (with intact protease ora subunit of a protease) or a host cell not engineered with thedeficient protease (or a subunit of a protease).

To quantitate the yield or amount of a protein (or polypeptide) producedby host cells, conventional methods such as immunoassay, highperformance liquid chromatography (HPLC), enzymatic activity,spectrophotometric techniques, SDS-PAGE and the like can be employed.

Pharmaceutical Compositions

A variety of different compositions such as pharmaceutical compositions,probiotic compositions, immunogenic compositions, and vaccines are alsoprovided by the present disclosure. These compositions may compriseproteins (or polypeptides) produced by the present host cells. Thesecompositions may comprise the present host cells (e.g., engineeredbacteria described herein).

In one embodiment, the present disclosure provides a pharmaceuticalcomposition comprising the following: (i) proteins (or polypeptides)produced by the present host cells; and (ii) a pharmaceuticallyacceptable carrier.

In another embodiment, the present disclosure provides a pharmaceuticalcomposition comprising the following: (i) the present host cells (e.g.,engineered bacteria described herein); and (ii) a pharmaceuticallyacceptable carrier.

Pharmaceutically acceptable carriers include any and all solvents,dispersion media, vehicles, coatings, diluents, antifungal agents,isotonic and absorption delaying agents, buffers, carrier solutions,suspensions, colloids, and the like. Pharmaceutically acceptablecarriers are well known to those of ordinary skill in the art, andinclude any material which, when combined with an active ingredient,allows the ingredient to retain biological activity. For instance,pharmaceutically acceptable carriers include, but are not limited to,water, buffered saline solutions (e.g., 0.9% saline), emulsions such asoil/water emulsions, and various types of wetting agents. Possiblecarriers also include, but are not limited to, oils (e.g., mineral oil),dextrose solutions, glycerol solutions, chalk, starch, salts, glycerol,and gelatin.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions, the type of carrierwill vary depending on the mode of administration. Compositions of thepresent invention may be formulated for any appropriate manner ofadministration, including for example, topical, oral, nasal,intravenous, intracranial, intraperitoneal, subcutaneous orintramuscular administration. In some embodiments, for parenteraladministration, such as subcutaneous injection, the carrier compriseswater, saline, alcohol, a fat, a wax or a buffer. In some embodiments,any of the above carriers or a solid carrier, such as mannitol, lactose,starch, magnesium stearate, sodium saccharine, talcum, cellulose,glucose, sucrose, and magnesium carbonate, are employed for oraladministration.

Compositions comprising such carriers may be formulated by conventionalmethods (see, for example, Remington's Pharmaceutical Sciences, 18thedition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990; andRemington, The Science and Practice of Pharmacy 20th Ed. MackPublishing, 2000).

The present invention also provides for a probiotic compositioncomprising the present engineered (recombinant) bacteria and/or proteins(or polypeptides) produced by the present host cells. The engineered(recombinant) bacteria may constitutively or inducibly express one ormore of proteins/polypeptides. In one embodiment, theproteins/polypeptides expressed are involved in carbohydrate metabolismand/or transport. In certain embodiments, the proteins can include aglycoside hydrolase, a galactokinase and a glucose/galactosetransporter. The gene of the protein/polypeptides may be under thecontrol of a constitutive or inducible promoter.

The probiotic composition may be administered enterally, such as oral,sublingual and rectal administrations.

The probiotic composition can be a food composition, a beveragecomposition, a pharmaceutical composition, or a feedstuff composition.

The probiotic composition may comprise a liquid culture. The probioticcomposition may be lyophilized, pulverized and powdered. As a powder itcan be provided in a palatable form for reconstitution for drinking orfor reconstitution as a food additive. The composition can be providedas a powder for sale in combination with a food or drink. The food ordrink may be a dairy-based product or a soy-based product. Typical foodproducts that may be prepared in the present disclosure may bemilk-powder based products; instant drinks; ready-to-drink formulations;nutritional powders; milk-based products, such as yogurt or ice cream;cereal products; beverages such as water, coffee, malt drinks; culinaryproducts and soups.

The composition may further contain at least one prebiotic. “Prebiotic”means food substances intended to promote the growth of probioticbacteria in the intestines.

The compositions can be, e.g., in a solid, semi-solid, or liquidformulation. Compositions can also take the form of tablets, pills,capsules, semisolids, powders, sustained release formulations,emulsions, suspensions, or any other appropriate compositions.

The present composition may be in the form of: an enema compositionwhich can be reconstituted with an appropriate diluent; enteric-coatedcapsules or microcapsules; powder for reconstitution with an appropriatediluent for naso-enteric infusion, naso-duodenal infusion orcolonoscopic infusion; powder for reconstitution with appropriatediluent, flavoring and gastric acid suppression agent for oralingestion; or powder for reconstitution with food or drink.

The composition may also contain conventional pharmaceutical additivesand adjuvants, excipients and diluents, including, but not limited to,water, gelatine of any origin, vegetable gums. ligninsulfonate, talc,sugars, starch, gum arabic, vegetable oils, polyalkylene glycols,flavouring agents, preservatives, stabilizers, emulsifying agents,buffers, lubricants, colorants, wetting agents, fillers, and the like.In all cases, such further components will be selected having regard totheir suitability for the intended recipient.

Immunogenic compositions are also provided by the present disclosure.For instance, the invention provides an immunogenic compositioncomprising a recombinant bacterium described herein and/or proteins (orpolypeptides) produced by the present host cells. In some embodiments,the immunogenic composition comprises recombinant bacteria, wherein apolypeptide expressed by the recombinant bacteria is an antigen orcomprises an antigen. In some embodiments, the recombinant bacterium inthe immunogenic composition expresses the polypeptide comprising theantigen at a level sufficient to induce an immune response to theantigen upon administration of the composition to a host (e.g., a mammalsuch as a human).

An additional application of the present host cells and methods iscreating synthetic organisms. Synthetic organisms can be used for avariety of purposes, including industrial enzyme production, biofuel andelectrical energy production, vaccine development, and anticancertherapies.

The following are examples of the present disclosure and are not to beconstrued as limiting.

Example 1

To produce recombinant NunΔN, E. coli BL21(DE3) with a clpP::kanmutation was transformed with a construct encoding SUMO-NunΔN. Afterpurification of SUMO-NunΔN, SUMO was cleaved using the SUMO proteaseUlp-1.

The engineered bacteria were constructed by P1 transduction of aclpP::kan from the KEIO collection into BL21 (DE3). This resulted in thegenotype of the bacteria being BL21(DE3) plus a clpP::kan mutation (akan insert in the clpP gene derived from the KEIO collection). The E.coli B strains obtained may be E. coli B BL21(DE3) clpP::kan or E. coliB BL21(DE3) ΔclpP. In one embodiment, the bacteria may be F-ompThsdS_(B) (r_(B)-m_(B)-) gal dcm (DE3) clpP::kan.

The P1 transduction may be conducted according to any suitable protocol.Current Protocols in Molecular Biology, 2007, Unit 1.17 E. coli GenomeManipulation by P Transduction. In one embodiment, BL21 (DE3) cells aregrown overnight in LB broth at 37° C. The cell pellet is harvested bycentrifugation and re-suspended in 10 mM MgSO₄. The cells are theninfected with the P1 bacteriophage grown on the KEIO strain (50 μlphage/100 μl cells). After 30 minutes at 37° C., 1 M sodium citrate isadded and the mixture is plated on LB+ kanamycin plates (50 ug/ml) at37° C.

Although a very low level of SUMO-NunDN (SUMO-NunΔN) was produced inBL21 (DE3), SUMO-NunDN was produced at a much higher level in BL21(DE3)with a clpP::kan mutation. As the BL21 (DE3) strain is also deficient inLon protease and OmpT protease, the BL21(DE3) with a clpP::kan mutationis deficient in Lon protease and OmpT protease, as well as ClpPprotease.

Specifically, 12 liters frozen stock of the BL21(DE3) cells with aclpP::kan mutation that were transformed with plasmids encodingSumo-NunΔN were thawed, and were induced with 0.5 mM IPTG when ODreached 0.8-1.0 (37° C.). The cells were then grown for about 4additional hours at a lower temperature (e.g., 30° C.) with 180 rpmshaking.

Sumo-NunΔN has a molecular weight of approximately 24.5 kDa, and a pI of6.49. After cleavage by the SUMO protease Ulp-1, the peptide NunΔN has amolecular weight of approximately 10.5 kDa, with a pI of 8.96.

In this experiment, Sumo-NunΔN is also polyhistidine tagged at theN-terminus. The sequence of polyhistidine-tagged Sumo-NunΔN is (SEQ IDNO:1):

MGHHHHHHHHHHSSGHIEGRHMASMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGSLESRDRRRIARWEKRIAYALKNGVTPGFNAIDDGPEYKINEDPMDKVDKALATPFPRDVEKIEDEKYEDVMHRVVNH AHQRNPNKKWS

Buffers

-   -   Lysis buffer: 50 mM Na-phosphate, pH7.9, 200 mM NaCl, 1 mM PMSF    -   Ni-A buffer: 50 mM Na-phosphate, pH7.9, 200 mM NaCl    -   Ni-B buffer: 50 mM Na-phosphate, pH7.9, 200 mM NaCl, 250 mM        imidazole    -   MonoS-A buffer: 50 mM Na-phosphate, pH7.5, 1 mM DTT    -   MonoS-B buffer: 50 mM Na-phosphate, pH7.5, 1M NaCl, 1 mM DTT    -   GF buffer: 1×TGE, 0.2M NaCl, 5 mM DTT

On day 1, cell pellet was resuspended in approximately 150 ml Lysisbuffer. Cells were lysed using French Press. The cell debris was removedby centrifugation at 15,000 rpm for 25 min at 4′ C. The centrifugationwas repeated once, and the supernatant was used in the subsequent stepsfor protein purification.

Two of 5 ml HiTrap IMAC columns (GE Healthcare Life Sciences) werecharged with Ni²⁺, and equilibrated with 3 column volumes (CV) of Ni-Band Ni-A buffer. The supernatant from the centrifugation was loaded ontothe equilibrated column at a flow rate of 1 ml/min. The column was firstwashed with 4% Ni-B buffer (10 mM imidazole), 8% Ni-B buffer (20 mMimidazole), 16% Ni-B buffer (40 mM imidazole), and then eluted withgradient 20%-70%, and increased to 1000/a Ni-B buffer over 12 CV. 5-mlfractions were collected.

Samples of different fractions were run on SDS-PAGE (FIG. 1B). Based onthe SDS-PAGE results, Fractions 8-13 were pooled for digestion by Ulp-1.

2 mM DTT and 0.9 mg Ulp-1 were added to 15 mg SUMO-NunΔN, and cleavagecarried out. Alternatively, 1 mg Ulp-1 can be added to 25 mg SUMO-NunΔN.

The sample was added into a dialysis bag with a 3.5 kDa MWCO(molecular-weight cutoff) and was dialyzed against 1 L MonoS Buffer at4° C. overnight.

An optional step after Ulp-1 cleavage is to centrifuge the sample for ashort-period of time (e.g., 15,000 rpm for 10 min at 4° C.).

On day 2, SDS-PAGE was run to confirm completion of the cleavage. Thesample was diluted 3-fold by the MonoS buffer, and loaded onto theequilibrated MonoS column (GE Healthcare Life Sciences). The MonoScolumn was washed with 10% MonoS-A buffer, and eluted with gradient10-100%/MonoS-B buffer over 18 CV. 4-ml fractions were collected.

Samples of different fractions were run on SDS-PAGE (FIG. 2B). Based onthe SDS-PAGE results, Fractions 6-10 were pooled (˜0.8 mg protein) andwere concentrated using 3 kDa Amicon (EMD Millipore).

The concentrate was injected to the equilibrated HiLoad SuperDex 75column (GE Healthcare Life Sciences) (FIG. 3A). FIG. 3B shows SDS-PAGEanalysis of samples of different fractions from the HiLoad SuperDex 75column. The purified peptides were >20 mg/mi. 50% glycerol was added toeach fraction to obtain a final concentration of 10% glycerol, and thepeptides were flash frozen in liquid nitrogen.

Results

Applied to the production of Sumo-NunDN, this technology demonstrated amarked improvement in efficiency as compared with the commerciallyavailable BL21(DE3) system. This genetically modified E. coli strainwith disrupted ClpP generated at least 10-fold greater proteinexpression levels (e.g., SUMO-NunDN) than commercial strains such as theBL21(DE3) system, which yields low to moderate levels of the protein.The ability of these genetically modified strains to improve yields ofunstable heterologous proteins makes this technology valuable forreducing the associated cost and time needed for protein production.

The scope of the present invention is not limited by what has beenspecifically shown and described hereinabove. Those skilled in the artwill recognize that there are suitable alternatives to the depictedexamples of materials, configurations, constructions and dimensions.Numerous references, including patents and various publications, arecited and discussed in the description of this invention. The citationand discussion of such references is provided merely to clarify thedescription of the present invention and is not an admission that anyreference is prior art to the invention described herein. All referencescited and discussed in this specification are incorporated herein byreference in their entirety. Variations, modifications and otherimplementations of what is described herein will occur to those ofordinary skill in the art without departing from the spirit and scope ofthe invention. While certain embodiments of the present invention havebeen shown and described, it will be obvious to those skilled in the artthat changes and modifications may be made without departing from thespirit and scope of the invention. The matter set forth in the foregoingdescription and accompanying drawings is offered by way of illustrationonly and not as a limitation.

Applicant incorporates herein by reference, in its entirety, the ASCIIfile denoted by the name Columbia Gottesman SEQ LIST_ST25-4.txt, havinga size of 2 KB, created on May 28, 2020. This Substitute Specificationcontains no new matter.

1. An engineered bacterium comprising at least one deficient protease,wherein the protease is Clp, ClpP, ClpQ (HslV), ClpAP, ClpXP, ClpAXP,ClpYQ, ClpA, ClpX, ClpY (HslU), or combinations thereof, and wherein thebacterium overexpresses a polypeptide at a level which is at least 2fold of the level of the polypeptide produced by a bacterium notengineered with the deficient protease.
 2. The engineered bacterium ofclaim 1, comprising deficient ClpP.
 3. The engineered bacterium of claim1, wherein a gene encoding the protease is knocked out or knocked downin the engineered bacterium.
 4. The engineered bacterium of claim 1,wherein a gene encoding the protease is mutated or deleted in theengineered bacterium.
 5. An engineered bacterium comprising a total orpartial deletion of at least one gene encoding a protease, wherein theprotease is Clp, ClpP, ClpQ (HslV), ClpAP, ClpXP, ClpAXP, ClpYQ, ClpA,ClpX, ClpY (HslU), or combinations thereof.
 6. The engineered bacteriumof claim 5, comprising a total deletion of a gene encoding ClpP.
 7. Theengineered bacterium of claim 5, further comprising a nucleic acidsequence encoding a polypeptide for overexpression.
 8. The engineeredbacterium of claim 1, further comprising deficient Lon, OmpT, FtsH, orcombinations thereof.
 9. The engineered bacterium of claim 2, furthercomprising deficient Lon and deficient OmpT.
 10. The engineeredbacterium of claim 1, wherein the bacterium belongs to the Escherichia,Methylomonas, Methylobacter, Methylococcus, Methylosinus, Salmonella,Erwina, Haematococcus, Rhodobacter, Myxococcus, Corynebacteria,Pseudomonas or Bacillus genus.
 11. The engineered bacterium of claim 1,wherein the bacterium is Escherichia coli.
 12. The engineered bacteriumof claim 11, wherein the Escherichia coli is E. coli BL21(DE3).
 13. Theengineered bacterium of claim 11, wherein the Escherichia coli is E.coli BL21.
 14. The engineered bacterium of claim 1, wherein thepolypeptide is an enzyme, a growth factor, a blood clotting factor, ahormone, or a transcription factor.
 15. The engineered bacterium ofclaim 1, wherein the polypeptide is a heterologous polypeptide.
 16. Theengineered bacterium of claim 1, wherein the bacterium overexpresses apolypeptide at a level which is at least 5 fold of the level of thepolypeptide produced by a bacterium not engineered with the deficientprotease.
 17. The engineered bacterium of claim 1, wherein the bacteriumoverexpresses a polypeptide at a level which is at least 8 fold of thelevel of the polypeptide produced by a bacterium not engineered with thedeficient protease.
 18. A method for overexpressing a polypeptide, themethod comprising the steps of: (a) culturing the engineered bacteriumof claim 1 to produce the polypeptide; and (b) isolating thepolypeptide.
 19. A method for overexpressing a polypeptide in bacteria,the method comprising the steps of: (a) transforming the engineeredbacterium of claim 5 with a nucleic acid sequence encoding thepolypeptide; (b) culturing the bacterium to produce the polypeptide; and(c) isolating the polypeptide.
 20. A method for expressing a polypeptidein bacteria, the method comprising the steps of: (a) engineering abacterial cell to decrease level and/or activity of at least oneprotease, wherein the protease is Clp, ClpP, ClpQ (HslV), ClpAP, ClpXP,ClpAXP, ClpYQ, ClpA, ClpX, ClpY (HslU), or combinations thereof; (b)transforming the engineered bacterial cell with a nucleic acid sequenceencoding the polypeptide; (c) culturing the bacterium to produce thepolypeptide; and (d) isolating the polypeptide.
 21. The method of claim20, wherein the polypeptide is a heterologous polypeptide.
 22. A methodfor producing the engineered bacterium of claim 1, wherein the proteaseis deleted by replacing the protease gene with a selection marker. 23.The method of claim 22, wherein the selection marker is an antibioticresistance gene.
 24. The method of claim 23, wherein the antibiotic iskanamycin, chloramphenicol, tetracyclin, ampicillin, vancomycin orerythromycin.
 25. The method of claim 23, wherein the antibiotic iskanamycin.